NOA36 Protein Contains a Highly Conserved Nucleolar Localization Signal Capable of Directing Functional Proteins to the Nucleolus, in Mammalian Cells

de Melo, Ivan S.; Jimenez-Nuñez, Maria D.; Iglesias, Concepción; Campos-Caro, Antonio; Moreno-Sanchez, David; Ruiz, Felix A.; Bolívar, Jorge

We constructed DNA encoding the NoLS of NOA36 fused with the Flag epitope and an exopolyphosphatase enzyme from yeast (scPPX1) that specifically hydrolyzes polyP into inorganic phosphate [41] . The expression of the protein in the nucleolus of transiently transfected cells was confirmed by confocal microscopy. Fig. 5A shows the immunolocalization of the Flag epitope, which was positive in at least 60% of the cells in all cases. Positive transfected cells with NoLS-scPPX showed a drastic reduction (less than 30% of the control) of the polyphosphate signal in the nucleolus (Fig. 5A, B ). In addition, when lacks NoLS, the positive transfected cells with scPPX show non-nucleolar localization at the same time as high amounts of nucleolar polyphosphate (Fig. 5A) . We confirmed the expression of the fused protein in extracts of transfected cells by western blot of the Flag epitope (Fig. 5C ). Only the cells transfected with the construct that included the fused protein showed a positive signal for the Flag epitope (Fig. 5C ).